The related retroviruses HTLV-L and bovine leukemia virus (BLV) both encode two regulatory proteins, termed Tax and Rex, that interact with specific cis-acting sequences to control virus transcription initiation and mRNA accumulation, respectively. The Rex protein is required for the cytoplasmic accumulation of viral mRNAs encoding structural proteins and interacts with sequence elements at the 3' end of the viral RNA. To better understand the molecular basis of Rex action, HTLV-I and BLV proviruses, Rex-deficient proviral mutants, and Rex expression plasmids were constructed. The cross-reactivities of BLV and HTLV-L Rex proteins and of the HIV-1 Rev protein were examined. The BLV and HTLV-I Rex proteins were interchangeable and the complementation was reciprocal. The Rex protein of HTLV-L but not of BLV could complement Rev-deficient HIV-1 expression. However, the Rev protein of HIV-1 was unable to complement Rex mutations in HTLV-I or BLV. The nonreciprocal actions of Rex and Rev proteins suggest that these factors interact directly with the cis-acting RNA elements of the viruses. The time course of HTLV-I gene expression after stimulation of a quiescent provirus or proviral mutants revealed that Rex has only a positive effect on RNA accumulation and does not negatively regulate expression. HTLV-I\BLV chimeric Rex proteins have been constructed to identify protein domains that interact with the RNA response elements. The ones that have been tested indicate that this domain is located in a region following the highly conserved N-terminal 20 amino acids. The BLV Rex protein has been expressed in a bacterial system and has been partially purified.